TOR regulates late steps of ribosome maturation in the nucleoplasm via Nog1 in response to nutrients

Abstract

The protein kinase TOR (target of rapamycin) controls several steps of ribosome biogenesis, including gene expression of rRNA and ribosomal proteins, and processing of the 35S rRNA precursor, in the budding yeast Saccharomyces cerevisiae. Here we show that TOR also regulates late stages of ribosome maturation in the nucleoplasm via the nuclear GTP-binding protein Nog1. Nog1 formed a complex that included 60S ribosomal proteins and pre-ribosomal proteins Nop7 and Rlp24. The Nog1 complex shuttled between the nucleolus and the nucleoplasm for ribosome biogenesis, but it was tethered to the nucleolus by both nutrient depletion and TOR inactivation, causing cessation of the late stages of ribosome biogenesis. Furthermore, after this, Nog1 and Nop7 proteins were lost, leading to complete cessation of ribosome maturation. Thus, the Nog1 complex is a critical regulator of ribosome biogenesis mediated by TOR. This is the first description of a physiological regulation of nucleolus-to-nucleoplasm translocation of pre-ribosome complexes.

Figure 1

TOR inactivation causes Nog1 accumulation in the nucleolus. (A) Nog1 is accumulated in the nucleolus upon rapamycin treatment. Cells of a strain expressing NOG1-GFP (SCU888) with plasmid pSCU632 (pDsRed-NOP1) were treated with rapamycin (200 ng/ml) for 30 min (Rap). GFP (green), DsRed (red) and DAPI (blue) signals were observed in cells. Nomarski images (DIC) were also recorded. Cells not treated with rapamycin were used as the control (Cont). (B) Enlarged images of rapamycin-treated cells.

Figure 2

Nutrient starvation causes Nog1 accumulation in the nucleolus. (A) Cells of a strain expressing NOG1-GFP (SCU888) with plasmid pSCU632 (pDsRed-NOP1) were transferred to carbon- (−C) or nitrogen-deprived (−N) medium for 30 min (−C). GFP (green), DsRed (red) and DAPI (blue) signals were observed in cells. (B) Nog1 is accumulated in the nucleolus upon carbon starvation. Cells of a strain expressing NOG1-GFP (SCU888) were transferred to carbon-deprived medium for 30 min (−C) and then re-supplemented with carbon in the presence (−C → +C, Rap) or absence (−C → +C) of rapamycin for 60 min. GFP and DAPI signals were observed in cells. (C) Nog1 is accumulated in the nucleolus upon nitrogen starvation. The same strain was transferred to nitrogen-deprived medium for 30 min (−N) and then re-supplemented with nitrogen in the presence (−N → +N, Rap) or absence (−N → +N) of rapamycin for 60 min. GFP and DAPI signals were observed in cells.

Figure 3

Pre-60S complex was accumulated in the nucleus as a result of Nog1 dysfunction. (A) Temperature-sensitive (ts) growth phenotype of nog1-ts mutants. The wild-type NOG1 (SCU801) and nog1 temperature-sensitive nog1-11 (SCU823), nog1-12 (SCU824) and nog1-13 (SCU825) cells were incubated on plates at 25°C for 3 days or at 37°C for 2 days. (B) Pre-60S complex was accumulated in the nucleus of nog1 temperature-sensitive mutants. Strains NOG1 (SCU783), nog1-11 (SCU823), nog1-12 (SCU824) and nog1-13 (SCU825), which harbor a plasmid expressing GFP-Rpl25 (pSCU615), were incubated at 37°C for 8 h and GFP signals were observed. (C) Strain nog1-11 (SCU823) harboring plasmid pGFP-RPL25 (pSCU615) was incubated at 37°C for 5 h and stained for GFP (Rpl25; green) and DAPI (blue).

Figure 4

Identification of components of the Nog1 complex. Cells of a strain expressing NOG1-TAP (SCU889) were harvested before (lane a) and 30 min after rapamycin (200 ng/ml) treatment (lane b). Nog1-associated proteins extracted from cells were purified according to the TAP purification protocol, and were separated by SDS–PAGE followed by silver staining. Wild-type strain TB50a (SCU425) was used as the negative control. Ribosomal (RP) and pre-ribosomal (Pre-RP) proteins were identified by mass spectrometric analysis.

Figure 5

Nop7 functions together with Nog1. (A) Overexpression of NOP7 suppresses thermosensitivity of nog1-ts strains. Strains nog1-11 (SCU823), nog1-13 (SCU825) and the wild-type NOG1 (SCU822) were transformed with a plasmid for overexpression of NOP7 (pSCU674) or with the empty vector (pSCU154). Serially diluted cells of each strain were spotted from the left to the right on YPAD plates. Plates were cultured at 25°C for 2 days or at 37°C for 1 day. (B) Overexpression of NOG1 suppresses thermosensitivity of nop7-td strains. nop7-td (SCU89) and the wild-type NOP7 (SCU88) strains were transformed with a plasmid for overexpression of NOG1 (pSCU388) or with the empty vector (pSCU379). Serially diluted cells of each strain were spotted from left to right on YPAD plates. Plates were cultured at 25°C for 2 days or at 34°C for 1 day. (C) Nop7 shows the STING phenotype. Cells of a strain expressing NOP7-GFP (SCU888) harboring a plasmid expressing DsRed-NOP1 (pSCU618) were treated with rapamycin (200 ng/ml, Rap) for 60 min. GFP (Nop7, green), RFP (Nop1, red) and DAPI (blue) signals were observed. Nontreated cells were used as the control (Cont).

Figure 6

Effects of rapamycin on localization of Rpl24 and Has1. (A) Strain SCU7 harboring a plasmid expressing RPL24-Myc13GFP (pSCU771) and DsRed-NOP1 (pSCU618) was treated with rapamycin (200 ng/ml, Rap) for 60 min, and GFP (Rlp24, green), RFP (Nop1, red) and DAPI (blue) signals were examined. Nontreated cells were used as the control (Cont). (B) Cells expressing HA2-HAS1 (SCU1285) were treated with rapamycin (200 ng/ml, Rap) for 60 min and stained for HA (Has1; green), Nop1 (red) and DAPI (blue).

Figure 7

Rapamycin-resistant NOG1-R1 strain is defective in STING. (A) Rapamycin-resistant growth of the NOG1-R1 mutant strain. Serially diluted cells of the wild-type NOG1 (SCU801) and NOG1-R1 (SCU1287) strains were spotted from left to right on YPAD plates containing various concentrations of rapamycin at 30°C for 3 days. (B) NOG1-R1 cells are defective in STING. NOG1 (SCU801) and NOG1-R1 (SCU1287) strains were treated with 50 ng/ml rapamycin for 60 min. Both types of cells expressed Nog1-HA3 and were stained for HA (Nog1; green) and DAPI (blue).

Figure 8

TOR regulates concentration of the Nog1 complex proteins. (A) NOG1 expression is upregulated by TOR. Cells (SCU7) were treated with rapamycin (200 ng/ml) or transferred to medium without a nitrogen or carbon source for 15 min. ACT1 was used as the loading control. (B) Changes in protein levels of Nog1, Nop7 and Nop1 after rapamycin treatment. NOP7-myc13 strain (SCU799) with plasmid pHAC33-NOG1 (pSCU387) was treated with rapamycin (200 ng/ml). Whole-cell extracts were subjected to Western blotting using anti-HA, anti-Myc, anti-Nop1 and anti-cyclin-dependent kinase (Cdk) antibodies. Cdk was used as the loading control. (C) Changes in protein levels of Nog1 after shut-off of NOG1 expression in the presence or absence of rapamycin. The wild-type cells (SCU425) harboring plasmid pGAL-HA-NOG1 (pSCU679) were pre-cultured in galactose-based medium for expressing HA-NOG1 and then supplemented with glucose for shut-off of NOG1 expression (at time 0). Rapamycin (200 ng/ml) was added simultaneously to cultures (+Rap). Cells not treated with rapamycin were prepared as the control (−Rap).

Figure 9

Rapamycin sensitivities and Nog1 protein levels of nog1-ts mutants. (A) Rapamycin-sensitive growth of nog1-11 and nog1-12. Serially diluted cells of NOG1 (SCU801), nog1-11, nog1-12 and nog1-13 strains were spotted from left to right on YPAD plates containing various concentrations of rapamycin at 30°C for 3 days. (B) Nog1 protein level of nog1-ts is decreased after shift to restrictive temperature in the presence of rapamycin. Each nog1-ts strain grown at 25°C was shifted to 37°C in the presence or absence of rapamycin (200 ng/ml) and the amount of Nog1 protein was determined by Western blotting using anti-HA antibody.